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Vitamin K exists in the food supply as phylloquinone, a plant-based form and as menaquinones (MKs), a collection of isoprenologues mostly originating from bacterial synthesis. Although multiple bacterial species used as starter cultures for food fermentations synthesize MK, relatively little is known about the presence and distribution of MK in the food supply and the relative contribution of MK to total dietary vitamin K intake. Dairy products may be a predominant source of dietary MK in many regions of the world, and there is recent interest in enhancing the MK content of dairy products through identification and selection of MK-producing bacteria in dairy fermentations. This interest is increased by emerging evidence that current dietary recommendations based on the classic role of vitamin K as an enzyme cofactor for coagulation proteins may not be optimal for supporting vitamin K requirements in extrahepatic tissues and that MK may have unique bioactivity beyond that as an enzyme cofactor. Observational studies have reported favorable associations between MK intake and bone and cardiovascular health. Although randomized trials have provided some evidence to support the beneficial effects of MK on bone, the evidence to date is not definitive, and randomized trials have not yet examined MK intake in relation to cardiovascular outcomes. Food production practices provide a means to enhance dietary MK availability and intake. However, parallel research is needed to optimize these production practices, develop comprehensive food MK content databases, and test hypotheses of unique beneficial physiological roles of MK beyond that achieved by phylloquinone.

Dairy propionibacteria, and especially Propionibacterium freudenreichii subsp. shermanii, are important in the food industry and biotechnology. Only a few investigations have focused on the complex physiology of this remarkable bacterium, while the physiology of dairy lactic acid bacteria has been extensively studied over the past decades. Here we report the progress of our whole genome sequencing project: 93% of the P. shermanii genome was assembled in 426 contigs with low overall redundancy. Our annotation strategy overlaps the sequence finishing step, thus improving it. Annotation of the incomplete genome is performed using ContigBrowser, a bioinformatic tool allowing data management, developed in our laboratory. This resulted in the detection of 2611 putative proteins (data May 2003). Our tool allows an expert annotation by manual verification and curation of functional protein categories after automatic assignment. Our genomic sequence analysis, combined with the already developed physiological, proteomic and metabolomic approaches, will allow researchers to explore the significant potentialities of dairy propionibacteria by providing a comprehensive view of the enzymes and metabolic pathways. This knowledge will allow researchers to explore more effective strategies to enhance the utility of this organism in manufacturing procedures or current industrial processes. Projet de s?quen?age du g?nome de Propionibacterium freudenreichii subsp. shermanii : ?tat d'avancement et premi?res analyses bioinformatiques. Les bact?ries propioniques laiti?res, et plus particuli?rement Propionibacterium freudenreichii subsp. shermanii, sont fortement utilis?es dans les secteurs de l'agroalimentaire et des biotechnologies. Les connaissances sur la physiologie complexe de cette bact?rie peu ordinaire sont faibles et parcellaires, contrairement aux bact?ries lactiques dont la physiologie a ?t? largement ?tudi?e depuis de nombreuses ann?es. Dans cet article, nous pr?sentons les avanc?es de notre projet de s?quen?age du g?nome complet de P. shermanii : 93% du g?nome (en 426 contigs) est d?j? disponible avec un faible taux global de recouvrement. Notre strat?gie d'analyse chevauche le proc?d? de finition du s?quen?age et l'am?liore. Nous avons mis en place ContigBrowser, un syst?me d'analyse bio-informatique d?di? ? l'annotation de g?nome incomplet et ? la gestion des donn?es. L'annotation nous a permis de mettre en ?vidence 2611 prot?ines putatives (donn?es mai 2003). L'analyse automatique des cat?gories fonctionnelles de prot?ines d'int?r?t a ?t? soumise ? un processus d'expertise par annotation manuelle. L'int?gration au sein d'une base de connaissances de l'analyse de notre s?quence g?nomique et des approches prot?omiques et m?tabolomiques, d?j? d?velopp?es au laboratoire, devrait fournir une vision globale des enzymes et des voies m?taboliques des bact?ries propioniques laiti?res. Cette vue int?gr?e du m?tabolisme pourrait faciliter l'exploration de potentialit?s significatives des bact?ries propioniques laiti?res. Cette base de connaissances devrait aussi permettre d'explorer de nouvelles strat?gies plus efficaces pour am?liorer l'utilisation de cette bact?rie dans les proc?dures de fabrication ou les proc?d?s industriels actuels.

Propionibacterium freudenreichiisubsp. shermaniiis known to prevent mutations caused by various agents such as N-methyl-N′-nitro-N-nitrosoguanidine, 9-aminoacridine, 4-nitro-quinoline-1-oxide and by UV radiation in both prokaryotic and eukaryotic cells. It was also shown to prevent or repair damage caused by H2O2or UV radiation in Salmonella typhimuriumand Escherichia coli, a characteristic previously designated as reactivative effect. In order to characterise this effect at the molecular level, we have purified the active component from a P. freudenreichiicell-free extract using a combination of ammonium sulfate precipitation, anion-exchange and size-exclusion chromatography. The isolated 35 kDa protein was then identified using both N-terminal and internal peptide sequencing as a cysteine synthase. The latter was localised in the P. freudenreichiiproteomic map. It is constitutively expressed but also clearly induced during adaptation to detergent and heat, but not acid, stresses. The biological meaning of cysteine synthase in the context of adaptation to oxidative and non-oxidative stresses is discussed.

Dairy propionibacteria, and especially Propionibacterium freudenreichii subsp. shermanii, are important in the food industry and biotechnology. Only a few investigations have focused on the complex physiology of this remarkable bacterium, while the physiology of dairy lactic acid bacteria has been extensively studied over the past decades. Here we report the progress of our whole genome sequencing project: 93% of the P. shermanii genome was assembled in 426 contigs with low overall redundancy. Our annotation strategy overlaps the sequence finishing step, thus improving it. Annotation of the incomplete genome is performed using ContigBrowser, a bioinformatic tool allowing data management, developed in our laboratory. This resulted in the detection of 2611 putative proteins (data May 2003). Our tool allows an expert annotation by manual verification and curation of functional protein categories after automatic assignment. Our genomic sequence analysis, combined with the already developed physiological, proteomic and metabolomic approaches, will allow researchers to explore the significant potentialities of dairy propionibacteria by providing a comprehensive view of the enzymes and metabolic pathways. This knowledge will allow researchers to explore more effective strategies to enhance the utility of this organism in manufacturing procedures or current industrial processes.

Lactobacillus delbrueckii ssp. bulgaricus is a lactic acid bacterium widely used in the dairy food industry. Since the industrial processes are a succession of constraints, it is essential to understand the behaviour of L. bulgaricus when facing usual stresses. The influence of heat stress was investigated on the viability of L. bulgaricus cells grown in a chemically defined medium. The susceptibility of cells to heat-shock was obvious only above 55 ?C. We investigated the acquisition of thermotolerance as a result of exposure to a moderate heat-shock, and the acquisition of a cross-stress-tolerance by exposure to a mild osmotic stress. When cells were submitted, before lethal temperature challenge (65 ?C), to a heat pre-treatment at 50 ?C or to a hyper-osmotic pre-treatment, the viability of cells increased. For the industrial strain RD 546, the addition of glycine betaine (GB) in 0.4 mol.L-1NaCl during the pre-treatment decreased the acquired thermotolerance, while GB alone enhanced cell viability. The thermotolerance of the type strain was not influenced by GB. We demonstrated that the stress tolerance induced by a moderate heat-shock was dependent on protein synthesis, while the effect of GB on RD 546 thermotolerance was independent of such biosynthesis. Thermotolerance acquired in presence of GB depends on a strain-dependant mechanism that differs from the mechanism involved after a moderate heat-shock. Stress thermique et thermotol?rance chez Lactobacillus delbrueckii ssp. bulgaricus . Lactobacillus delbrueckii ssp. bulgaricus est une bact?rie lactique largement utilis?e en industrie alimentaire. Les proc?d?s industriels ?tant une succession de contraintes, il est essentiel de conna?tre le comportement de L. bulgaricus face aux stress rencontr?s. L'influence du stress thermique sur la viabilit? de 2 souches de L. bulgaricus, cultiv?es en milieu chimiquement d?fini, a ?t? ?tudi?e. La viabilit? des cellules n'est affect?e qu'au-del? de 55 ?C. Les cellules acqui?rent une thermotol?rance vis-?-vis d'un choc thermique ? 65 ?C durant 10 min, c'est-?-dire une viabilit? accrue, apr?s exposition ? un pr?traitement thermique mod?r? ? 50 ?C ou un pr?traitement hyperosmotique. Pour la souche industrielle RD 546, l'addition de glycine b?ta?ne (GB) dans le milieu de pr?traitement contenant 0,4 mol.L-1NaCl diminue la thermotol?rance acquise, tandis que la glycine b?ta?ne seule en tant que pr?traitement avant le choc l?tal augmente la viabilit? cellulaire. La thermotol?rance induite par un pr?traitement thermique mod?r? est d?pendante de la synth?se prot?ique, tandis que l'effet de la GB sur la thermotol?rance est ind?pendant de biosynth?se. La thermotol?rance acquise en pr?sence de GB n?cessite un m?canisme souche-d?pendant distinct par rapport ? celui impliqu? lors d'un pr?traitement thermique.

Lactobacillus delbrueckii ssp. bulgaricus is a lactic acid bacterium widely used in the dairy food industry. Since the industrial processes are a succession of constraints, it is essential to understand the behaviour of L. bulgaricus when facing usual stresses. The influence of heat stress was investigated on the viability of L. bulgaricus cells grown in a chemically defined medium. The susceptibility of cells to heat-shock was obvious only above 55 $^{\circ}$C. We investigated the acquisition of thermotolerance as a result of exposure to a moderate heat-shock, and the acquisition of a cross-stress-tolerance by exposure to a mild osmotic stress. When cells were submitted, before lethal temperature challenge (65 $^{\circ}$C), to a heat pre-treatment at 50 $^{\circ}$C or to a hyper-osmotic pre-treatment, the viability of cells increased. For the industrial strain RD 546, the addition of glycine betaine (GB) in 0.4 mol.L$^{-1}$ NaCl during the pre-treatment decreased the acquired thermotolerance, while GB alone enhanced cell viability. The thermotolerance of the type strain was not influenced by GB. We demonstrated that the stress tolerance induced by a moderate heat-shock was dependent on protein synthesis, while the effect of GB on RD 546 thermotolerance was independent of such biosynthesis. Thermotolerance acquired in presence of GB depends on a strain-dependant mechanism that differs from the mechanism involved after a moderate heat-shock.

This study used in vivo13C NMR spectroscopy to directly examine bidirectional reactions of the Wood–Werkman cycle involved in central carbon metabolic pathways of dairy propionibacteria during pyruvate catabolism. The flow of [2-13C]pyruvate label was monitored on living cell suspensions of Propionibacterium freudenreichiisubsp. shermaniiand Propionibacterium acidipropioniciunder acidic conditions. P. shermaniiand P. acidipropionicicells consumed pyruvate at apparent initial rates of 161 and 39μmol min−1g−1(cell dry weight), respectively. The bidirectionality of reactions in the first part of the Wood–Werkman cycle was evident from the formation of intermediates such as [3-13C]pyruvate and [3-13C]malate and of products like [2-13C]acetate from [2-13C]pyruvate. For the first time alanine labeled on C2 and C3 and aspartate labeled on C2 and C3 were observed during [2-13C]pyruvate metabolism by propionibacteria. The kinetics of aspartate isotopic enrichment was evidence for its production from oxaloacetate viaaspartate aminotransferase. Activities of a partial tricarboxylic acid pathway, acetate synthesis, succinate synthesis, gluconeogenesis, aspartate synthesis, and alanine synthesis pathways were evident from the experimental results.

Propionibacterium starters are subjected to many stresses during Swiss-type cheese manufacture. Among the phenomena which exert strong effects on Propionibacterium growth, osmotic stress influences the cell activity, the spatial cell distribution in the loaf and also drastically influences cheese ripening. In a chemically defined medium (M63), the generation time of Propionibacterium freudenreichii subsp. shermanii CIP 103027 was doubled by 0.3 mol-L-1sodium chlo-ride and at 0.7 mol-L-1, growth was almost totally inhibited. Growth in rich medium was more resistant to hyperosmotic condition.. 13C Nuclear magnetic resonance (NMR) spectroscopy was used to investigate the intracellular solute pools in living cell suspensions and in hydroalcoholic extracts. Glutamate and trehalose were the main osmolytes found in the intracellular fraction of the cells in M63 containing 0.5 mol-L-1NaCl. Addition of glycine-betaine, dimethylsulfoniopropionate or dimethylsulfonioacetate to this medium partially restored the growth. These efficient osmoprotectors were accumulated by the stressed cells. Moreover, glutamate and trehalose synthesis were also enhanced. In a rich medium. P. shermanii cells responded to changes in osmolarity by increasing the concentrations of specific solutes, and especially glycine-betaine, in order to maintain a constant turgor pressure. Implications of such behavior in cheese technology are discussed. ? Inra / Elsevier, Paris.Au cours de la fabrication fromag?re, les bact?ries propioniques sont soumises ? diff?rents stress. Parmi ceux-ci figure le stress osmotique, r?sultat du saumurage, qui influence consid?rablement la croissance des cellules mais aussi leur distribution au sein de la meule et donc conditionne l'affinage du fromage. Dans un milieu chimiquement d?fini, le temps de g?n?ration de Propionibacterium freudenreichii subsp. shermanii CIP 103027 double en pr?sence d'une concentration de chlorure de sodium de 0,36 mol-L-1. ? 0,7 mol-L-1Ia croissance est totalement inhib?e. Dans un milieu riche, les cellules sont plus r?sistantes ? l'augmentation de la pression osmotique. Le tr?halose et le glutamate sont les principaux osmolytes accumul?s lors d'une croissance en M63 + 0,5 mol-L-1NaCl. Une addition de glycine-b?ta?ne, de dim?thylsulfoniopropionate ou de dim?thylsulfonioac?tale au M63 restaure partiellement la croissance. Mais une addition de tr?halose n'apporte pas d'effet protecteur aux cellules. Une ?tude par r?sonance magn?tique nucl?aire du carbone 13 montre que ces osmolytes sont accumul?s par les cellules. Lors de culture en milieu riche (YEL). les cellules de Propionibacterium freudenreichii subsp. shcnnanii accumulent de la glycine-b?ta?ne pr?lev?e dans le milieu. Les implications potentielles du comportement de Propionibacterium freudenreichii subsp. shermanii en technologie fromag?re sont discut?e.? Inra / Elsevier, Paris.

Bacteriocins are natural proteinaceous antimicrobial substances produced by microorganisms like lactic acid bacteria to dominate the other microflora. Their use in the food industry as natural preservatives is tightly linked to the emergence of efficient and inexpensive production and purification procedures. The production of an anti-Listeria bacteriocin, named Divercin, by Carnobucterium divergens, has been studied in batch, continuous culture and with viable cells immobilized in alginate gel beads retained in a plug flow bioreactor. The latter System gave the highest volumetric productivity with more than 105AU L-1h-1, which was one hundred times the pro-ductility of the batch system. A new simple (two steps) bacteriocin purification method was developed based on Triton -114 phase partitioning. The pilot production of bacteriocin can then be achieved allowing the exploration of the impact of such additions on the microflora of foodstuffs. ? Inra / Elsevier, Paris.Les bact?riocines sont des substances naturelles de nature prot?ique, produites par des micro-organismes et qui pr?sentent une activit? bact?ricide ou bact?riostatique. L'emploi, dans les industries alimentaires, de bact?riocines produites par des bact?ries lactiques n?cessite des m?thodologies de production et de purification extrapolables afin de r?aliser des exp?rimentations pilotes. La production d'une bact?riocine active contre Listeria. la Divercine V41, par des cellules enti?res de Carnobacterium divergens V41, a ?t? ?tudi?e en culture discontinue, en culture continue avec des cellules libres et ? l'aide de cellules immobilis?es dans des billes d'alginate de calcium plac?es dans un r?acteur piston. Les productivit?s et les concentrations atteintes en bact?riocines ont ?t? compar?es. Les cellules immobilis?es pr?sentent une bonne productivit? avec plus de 105UA L-1h-1, que l'on peut avantageusement comparer ? celle du syst?me discontinu : 2,8.10-1UA.L-1.h-1. Une m?thode de purification simple, rapide, et bon march?, fond?e sur l'emploi d'un d?tergent, a ?t? d?velopp?e. La production pilote de bact?riocine peut donc ?tre raisonnablement envisag?e et des ?tudes d'impact, au sein des aliments r?alis?es. ? Inra / Elsevier, Paris.

Lactose of sweet whey permeate was converted into sodium lactate byLactobacillus helveticus. To increase the, productivity of the lactic acid fermentation and to reduce the amounts of effluents, the bioreactor was coupled with an ultrafiltration module and an electrodialysis unit. Without the electrodialyzer, with total cell recycling and at a dilution rate of 0.88 h−1, a cellular concentration of 64 gl−1and a productivity of 22 gl−1h−1were obtained. When the electrodialysis unit is coupled, the outlet concentration of lactate was stabilized at 85±5 gl−1.

The present work reports the survival capacity of a strain of Brevibacterium linens isolated from a French camembert cheese and the ensuing changes in cell composition. Exponentially growing cells were harvested, washed and resuspended with shaking in pH 8.0 buffer at 21°C in the absence of a carbon source. The viability of this strain, assessed with slide cultures, is much less than that of coryneform bacteria isolated from soil samples, even though no cell lysis was detected. Intracellular RNA was rapidly consumed during the first few days although magnesium levels remained high. The quantity of DNA initially increased by 17% within 24 h and then remained stable during the 30 days of the experiment. During the same period, absorbance of the medium at 260 nm reached 2 absorbance units. Reserve polysaccharides in this strain are less abundant than in Arthrobacter and were rapidly consumed. Proteolysis was regular and thus maintained a pool of free amino acids which was greater than 60% of the initial value. There was a parallel accumulation of ammonia in the medium. Catalase activity decreased regularly during the first 80 h whereas the quantity of Adenosine-5'-triphosphate (ATP) dropped by 47% in 10 h, stabilizing at less than 10% of its initial value. Cell respiration declined very rapidly and was very low after 24 h.

ABSTRACTEmerging evidence indicates maternal microbiota as one major reservoir for pioneering microbes in infants. However, the global distinct and identical features of mother–infant gut microbiota at various taxonomic resolutions and metabolic functions across cohorts and potential of infant microbial prediction based on their paired mother’s gut microbiota remain unclear. Here, we analyzed 376 mother–infant dyads (468 mother and 1024 infant samples) of eight studies from six countries and observed higher diversity at species and strain levels in maternal gut microbiota but not their metabolic functions. A number of 290 species were shared in at least one mother–infant dyad, with 26 species (five at strain level) observed across cohorts. The profile of mother–infant shared species and strains was further influenced by delivery mode and feeding regimen. The mother-sourced species in infants exhibited similar strain heterogeneity but more metabolic functions compared to other-sourced species, suggesting the comparable stability and fitness of shared and non-shared species and the potential role of shared species in the early gut microbial community, respectively. Predictive models showed moderate performance accuracy for shared species and strains occurrences in infants. These generalized mother–infant shared species and strains may be considered as the primary targets for future work toward infant microbiome development and probiotics exploration.